Selection Of Aptamer And Its Application To Rapid Detection Methods Of Thiamethoxam

Thiamethoxam is a typical second-generation neonicotinoid insecticide,which is widely used in agricultural production.Due to its gradual exposure to hazards to non-target organisms and humans,the problem of residues in the environment and agricultural products is more and more concerned.Vegetables are the main economic crops in our country.Due to the short market cycle and complex sample matrix,traditional detection methods cannot meet the needs of rapid screening and detection.Therefore,there is an urgent need to establish a rapid and sensitive analytical method to detect thiamethoxam residues in vegetables.Aptamers are single-stranded oligonucleotides screened in vitro by systematic evolutionof ligands by exponential enrichment（SELEX）.With high affinity and specificity for their targets,they are ideal bio-recognition elements and show great potential in rapid detection.In this paper,graphene oxide（GO）-SELEX technology was used to screen aptamers for thiamethoxam,and the sequences obtained by screening were further truncated to obtain truncated sequences with higher affinity.At the same time,the binding mechanism of aptamer and thiamethoxam was explored by circular dichroism spectroscopy.On this basis,the biosensor was constructed to realize the rapid detection of thiamethoxam using the obtained aptamer as a recognition element,combined with colorimetric and electrochemical techniques.The constructed sensor has been successfully applied to actual vegetable samples,which provides a good foundation for the rapid detection of thiamethoxam residues in the future.The specific research was as follows:（1）Aptamers for thiamethoxam were screened using the non-immobilized GO-SELEX technology.Using GO to adsorb ssDNA to achieve rapid separation of thiamethoxam-bound and unbound ssDNA in the library.After 9 rounds of positive selection and counter selection to enrich the library,33 sequences were obtained by cloning and sequencing.5 representative sequences were selected as candidate sequences by the primary structure homology and secondary structure difference analysis of the obtained 33sequences.（2）Identification of candidate sequences obtained by screening using colorimetry.The affinity of candidate sequences was determined,among which,seq.20 exhibited a lower dissociation constant and therefore had a higher affinity.Then,based on secondary structure analysis,seq.20 was further truncated to obtain aptamers seq.20-1 and seq.20-2with higher affinity,and seq.20-2 had only 37 bases.Using circular dichroism spectroscopy to explore the binding mechanism of thiamethoxam and aptamer,it was found that thiamethoxam could induce the conformational change of aptamer to form a hairpin structure and then specifically bind to thiamethoxam to form a thiamethoxam-aptamer complex.In addition,a colorimetric aptasensor based on gold nanoparticles was established to verify the performance of the original aptamer and the truncated aptamer in the actual detection of thiamethoxam.Compared with the original aptamer,the sensor constructed by the truncated aptamer had a wider detection range and lower detection limit.（3）An ultrasensitive electrochemical aptasensor for the detection of thiamethoxam was constructed using a composite material of porous reduced graphene oxide and Au@Fe₃O₄shell-core structure.Porous reduced graphene oxide was prepared by etching method,and the formed porous structure effectively overcame the problem of easy aggregation of graphene,and had a large specific surface area.The Au@Fe₃O₄shell-core structure organically combined the easy surface modification properties of AuNPs with the large specific surface area of Fe₃O₄,which provided a good loading interface for the immobilization of bio-recognition elements.Furthermore,porous reduced graphene oxide and Au@Fe₃O₄exhibited a remarkable synergistic effect,effectively promoting electron transfer and improving the electrochemical performance.In addition,the truncated aptamer seq.20-2 obtained from the previous two chapters was used as the recognition element to realize the specific recognition of thiamethoxam.Under the optimal experimental conditions,the sensor exhibited a wide linear range（0.1 ng/m L-1000 ng/m L）and low limit of detection（LOD=0.03 ng/m L）,and had a good recovery（91.2%-109.6%）in vegetable samples.